Interview with Henri-Pierre FIEROBE

On May 30, 2022, the iGEM team went to interview M. Henri-Pierre Fierobe, Research Director in the team "CELLULOSOMES AND DEGRADATION OF PLANT POLYMERS" in the Laboratory of Bacterial Chemistry. We had questions to ask him about the cellulosome since we wanted to modify the cellulosome to make a "plasticosome". Our questions were therefore the following :

• Primers used for amplification ? From gDNA ?
• How to purify the cellulosome ? Can it be produced in large quantities in E.coli ?
• By which secretion systems is it secreted ?
• Which dockerin sequences to use ? Can it be produced alone? Protocol to fuse our dockerin with our laccase ?

Dr Fierobe answered us, in his laboratory with his team, that they have developed an artificial mini cellulosome that is to say that it has 3 cohesins of different species that each recognize a different dockerin. This allows a better organization of the cellulosome.

Concerning secretion, he pointed out that there were secretion problems in E.coli. Indeed, the cellulosomes are secreted by anaerobic Gram + organisms like Ruminiclostridium cellulolyticum (Microorganism subject of study of Dr.Fierobe's team) except that E.coli is an aerobic Gram - bacteria so he told us that the cellulosome was blocked in the periplasm.

He therefore advised us several possibilities such as the E.coli curli system which, under stress, produces filaments to which parts of the cellulosome could be grafted in C-term, except that the graft is irreversible. Alternatively, try the Tat machinery for secretion in E.coli or have the cellulosome secreted by Bacillus subtilis. For cloning, do classical or Slic cloning.

Interview with Thierry TRON

On May 31, 2022, the iGEM team went to interview M. Thierry Tron, CNRS Research Director : Institute of Molecular Sciences of Marseille. We had questions to ask him about the laccase enzyme, its structure, its activity and its plausible use to degrade microplastics".

Dr Tron took the time to exchange with and explained to us the enzyme structure, how the copper centers were organized and how the potential plays a key role in the oxidation and reduction reaction catalyzed by the laccase. Originally laccase catalyzes the oxidation of different phenolic compounds or aromatic amines, as microplastic aren’t strictly the ideal substrate for laccase and considering their size the use of a mediator could be helpful for our project. A mediator is a small molecule that is more easily accessible in the environment and whose oxidation by the laccases generates unstable free radicals which in turn will diffuse into the microplastic’s structure and cause bond breaks, making the degradation of microplastics possible.
However, our project is not suitable for the use of mediators and we later decided with all the team reunited to try to design the laccase so that its oxidation site could be more accessible to microplastics.

As we wanted to try to produce and test both bacterial and fungal laccase, Dr Tron pointed out to us that we should take into account that fungal laccase are glycosylated which make its production difficult in E. Coli.

Please find in the following link one of their articles DOI: 10.1002/cplu.201700030 focusing on probing the surface of a fungal Laccase for clues towards the design of Chemo-Enzymatic Catalysts which present a strategy to control the orientation of a graft at the surface on the protein.